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| United States Patent |
5,747,029
|
|
Walker
, et al.
|
May 5, 1998
|
Control of weeds with a fungal pathogen derived from M. verrucaria
Abstract
A method for the biological control of various weeds such as sicklepod
using the fungus Myrothecium verrucaria. In a typical application, conidia
of the fungus are applied with liquid surfactant to the weeds in amounts
effective to produce typical plant lesions which kill or suppress, and
thus control, the weeds. In another embodiment, phytotoxin produced by the
fungus is used to control the weeds. In still another embodiment a
synergistic combination of M. verrucaria and the fungus Alternaria cassiae
is used to control sicklepod and in a fourth embodiment phytotoxin
produced by M. verrucaria is used synergistically with A. cassiae to
control sicklepod. A strain of M. verrucaria is on deposit with the
Department of Biological Sciences, Louisiana Tech University in Ruston,
La. and with the patent collection of the International Mycological
Institute in Surrey, UK and has been assigned the number IMI 368023.
| Inventors:
|
Walker; Harrell L. (1171 Highway 3072, Ruston, LA 71270);
Tilley; Anthony M. (2567 Doc Steed Rd., Minden, LA 71055)
|
| Appl. No.:
|
512093 |
| Filed:
|
August 7, 1995 |
| Current U.S. Class: |
424/93.5; 424/93.3; 435/254.1; 435/911 |
| Intern'l Class: |
A01N 063/00 |
| Field of Search: |
435/254.1,911
424/93.5,93.3
|
References Cited
U.S. Patent Documents
| 4390360 | Jun., 1983 | Walker | 71/79.
|
| 4419120 | Dec., 1983 | Walker | 71/79.
|
| 4636386 | Jan., 1987 | Anderson et al. | 424/93.
|
| 4643756 | Feb., 1987 | Cardina et al. | 71/79.
|
| 4755207 | Jul., 1988 | Bannon | 71/79.
|
| 4767441 | Aug., 1988 | Walker et al. | 71/79.
|
| 4776873 | Oct., 1988 | Caulder et al. | 71/79.
|
| 4808207 | Feb., 1989 | Gotlieb et al. | 71/73.
|
| 4871386 | Oct., 1989 | Riley | 71/79.
|
| 4902333 | Feb., 1990 | Quimby, Jr. | 71/79.
|
| 5074902 | Dec., 1991 | Connick, Jr. et al. | 71/79.
|
| 5314691 | May., 1994 | Coffey et al. | 424/93.
|
Other References
Phytopathology,vol. 84:10, 1994 pp. 1136-1137, Control of Carduus
Acanihoides With Myrothecium Verrucaria in the Greenhouse In the Absence
of Dew Yang. Abstract #562.
Detrich IBG News, May 1995, vol. 4, No. 1, pp. 7-8 "Classical Biological
Control of Weeds With Pathogens".
Plant Diseases Oct., 1995, vol. 79 No. 10 pp. 994-997, Host Range
Determination of Myrothecium Verrucaria from Leafy Spurge-Yang et al.
Plant Diseases Oct., 1995,vol. 79 No. 10,pp. 998-1002 Factors Influencing
Pathogenicity of Myrothecium Verrucaria Isolated From Euphorbia escla on
Species of Euphorbia-Yang et al.
|
Primary Examiner: Sayala; Chhaya D.
Attorney, Agent or Firm: Harrison; John M., Brown; Randall C., Matos; Rick
Claims
Having described my invention with the particularity set forth above, what
is claimed is:
1. A method for the biological control of weeds, comprising the steps of
culturing a herbicidal effective amount of conidia of the fungus
Myrothecium verrucaria (Alb. and Schwein.) Ditmar ex Fr. on a suitable
growth medium; harvesting said conidia; and inoculating the weeds with a
herbicidal effective amount of said conidia of the fungus Myrothecium
verrucaria (Alb. and Schwein.) Ditmar ex Fr., wherein said weeds are
inoculated with said conidia by applying said conidia to said weeds in a
liquid surfactant.
2. The method of claim 1 wherein said weeds are inoculated with a mixture
of conidia of the fungus Myrothecium verrucaria (Alb. and Schwein.) Ditmar
ex Fr. and conidia of the fungus Alternaria cassiae (Juriar and Khan) in a
liquid surfactant.
3. The method of claim 1 wherein said surfactant is selected from the group
consisting of a silicone-polyether copolymer spray adjuvant, oxysorbic (20
POE) (polyoxyethylene sorbitan mono oleate) and nonoxynol (9 to 10 POE)
(.alpha.-›.rho.-nonylphenyl-.omega.-hydroxypoly(oxyethylene)!).
4. A method for the biological control of a variety of weeds, comprising
the steps of culturing a herbicidal effective amount of conidia of the
fungus Myrothecium verrucaria (Alb. and Schwein.) Ditmar ex Fr. on a
suitable growth medium; harvesting said conidia; rinsing said conidia with
a liquid surfactant; and inoculating the weeds with a mixture of said
liquid surfactant and said conidia.
5. The method of claim 4 further comprising the step of mixing the fungus
Alternaria cassiae (Juriar and Khan) with the fungus Myrothecium
verrucaria (Alb. and Schwein.) Ditmar ex Fr. prior to said inoculating of
the weeds.
6. The method of claim 4, wherein said surfactant is selected from the
group consisting of a silicone-polyether copolymer spray adjuvant,
oxysorbic (20 POE) polyoxyethylene sorbitan mono oleate and nonoxynol (9
to 10 POE) ›.alpha.-(.rho.-nonylphenyl)-.omega.-hydroxypoly
(oxyethylene)!.
7. A method for the biological control of weeds growing with crop plants,
comprising the steps of inoculating the weeds with a herbicidal effective
amount of conidia of the fungus Myrothecium verrucaria (Alb. and Schwein.)
Ditmar ex Fr. in a liquid surfactant and applying said conidia of the
fungus and said liquid surfactant to the weeds beneath the canopy of the
crop plants.
8. The method of claim 7 wherein said surfactant is selected from the group
consisting of a silicone-polyether copolymer spray adjuvant, oxysorbic (20
POE) (polyoxyethylene sorbitan mono oleate) and nonoxynol (9 to 10 POE)
(.alpha.-›.rho.-nonylphenyl-.omega.-hydroxypoly(oxyethylene)!).
9. A method for the biological control of sicklepod comprising the step of
inoculating the sicklepod with a herbicidal effective amount of the fungus
Myrothecium verrucaria (Alb. and Schwein.) Ditmar ex Fr. in a liquid
surfactant.
10. The method of claim 9 wherein said sicklepod is inoculated with a
mixture of the fungus Myrothecium verrucaria (Alb. and Schwein.) Ditmar ex
Fr. and the fungus Alternaria cassiao (Juriar and Khan) in a liquid
surfactant.
11. The method of claim 9 wherein said surfactant is selected from the
group consisting of a silicone-polyether copolymer spray adjuvant,
oxysorbic (20 POE) (polyoxyethylene sorbitan mono oleate) and nonoxynol (9
to 10 POE) (.alpha.-›.rho.-nonylphenyl-.omega.-hydroxypoly(oxyethylene)!).
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to bioherbicides for controlling pest plants such as
weeds and more particularly, to a method for the biological control of a
variety of pest plants, including sicklepod, using the fungus Myrothecium
verrucaria. Typically, in a first embodiment a selected concentration of
conidia in a mixture of distilled water and liquid surfactant such as
SILWET L-77 (trademark), is applied to the pest plants to produce typical
lesions which kill or suppress, and thus control, the plants. In a second
embodiment, phytotoxin is rinsed from the conidia by subjecting the
conidia to sequential centrifugation cycles, and applied to the plants
using distilled water or a mixture of distilled water and a surfactant. In
a third embodiment, a synergistic combination of M. verrucaria and the
fungus Alternaria cassiae in a mixture of distilled water and surfactant
is applied to the plants to achieve enhanced control of the plants, and in
a fourth embodiment, phytotoxin extracted from M. verrucaria is used in
combination with Alternaria cassiae in a mixture of distilled water and
surfactant to synergistically control the pest plants.
Weeds present a tremendous problem to farmers throughout the world, causing
an estimated 10-12% loss of value for agricultural products in the United
States, the most recent estimate being $20 billion annually, according to
McWhorter, C. G. ›1984! Weed Science, 32:850-855. Chemical pesticides are
commonly used to control weeds in agricultural crops, but concern over
environmental damage caused by these pesticides has recently elicited
societal pressures to replace the chemical pesticides with alternative
control methods. One area of active research in this area involves the use
of plant pathogens, including both bacteria and fungi, to attack and kill
pest plants in agricultural crops.
A major constraint to commercial development of a plant pathogen as a
biological herbicide can be selectivity. A pathogen that controls only one
weed species in one type of crop does not have the same market potential
as a pathogen that controls several important weed species in different
types of crops. It has surprisingly been found that the fungus Myrothecium
verrucaria is effective in controlling multiple varieties of weeds in
several different types of important agricultural crops. In addition to
controlling sicklepod, the M. verrucaria utilized in this invention is
effective in controlling pigweed, spurred anoda, jimsonweed, hemp
sesbania, and other susceptible weeds, but does not harm Bermudagrass and
Centipedegrass and therefore may also be used to control weed growth in
some residential and commercial lawns and turf. It has also surprisingly
been found that conidia of M. verrucaria or phytotoxin produced by M.
verrucaria may be applied to the weeds in combination with conidia of the
fungus Alternaria cassiae, to enhance the pathogenic action of A. cassiae
on sicklepod.
2. Description of the Prior Art
Several methods are known in the art for using microorganisms to control
weeds and other pest plants. As disclosed in U.S. Pat. No. 3,999,973, to
Daniel, et al. the anthracnose fungus Colletotrichum gloeosporioides has
been used to control the weed northern jointvetch and another strain of
this fungus has been used to control winged waterprimrose. Colletotrichum
malvarum has been used to control prickly sida. These three pathogens have
been combined to control all three target weeds at once. In other
experimental work the fungus Alternaria macrospora has been used to
control spurred anoda (Anoda cristata), Weed Science, H. L. Walker, 1981,
Vol. 29, pp. 505-507.
Research activity involving Myrothecium verrucaria is noted on page 8 of
the IBG News, May, 1995 issue, Vol. 4 No. 1. My U.S. Pat. No. 4,390,360,
dated Jun. 28, 1983, describes "Control of Sicklepod, Showy Crotalaria and
Coffee Senna With A Fungal Pathogen" using a specific host strain of the
fungus Alternaria cassiae to produce typical weed lesions which kill or
suppress the respective weeds. My U.S. Pat. No. 4,419,120, dated Dec. 6,
1983, discloses "Control of Prickly Sida, Velvetleaf and Spurred Anoda
With Fungal Pathogens" using a specific host strain of the fungus Fusarium
lateritium to kill or suppress the respective weeds. U.S. Pat. No.
4,715,881, dated Dec. 29, 1987, to Andersen, et al., details "Control of
Eastern Black Nightshade With a Fungal Pathogen" using a strain of
Colletotrichum coccodes which is pathogenic toward eastern black
nightshade (Solanum ptycanthum). My U.S. Pat. No. 4,718,935, dated Jan.
12, 1988 and U.S. Pat. No. 4,767,441, dated Aug. 30, 1988, describe a
"Method For The Preparation of Mycoherbicide-Containing Pellets"
characterized by alginate gel pellets containing living fungus capable of
producing conidia when exposed to sufficient light and moisture. U.S. Pat.
No. 4,724,147, dated Feb. 9, 1988, to James J. Marois, et al. and U.S.
Pat. No. 4,818,530, dated Apr. 4, 1989, also to James J. Marois, et al.
both detail the "Preparation of Pellets Containing Fungi for Control of
Soilborne Diseases", in which fungi are first selected and grown for a
time sufficient to produce inoculum. The fungal propagules are harvested,
homogenized and diluted with sodium alginate solution. Pelletization is
then accomplished by dropwise addition of the fungal propagule-alginate
mixture into a solution of calcium chloride or calcium gluconate. The
resulting alginate gel pellets containing living fungi can then be dried
and used to inoculate agricultural fields infested with soilborne plant
diseases. U.S. Pat. No. 5,192,541, dated Mar. 9, 1993, to Steven D.
Savage, et al. describes "Weed-Killing Xanthomonas campestris", in which
novel microorganisms useful in controlling unwanted grasses and other
weeds are discovered through a unique process which involves isolating
plant pathogens from asymptomatic plants. U.S. Pat. No. 5,393,728, dated
Feb. 28, 1995, to Charudattan, et al. details a "Broad Spectrum
Bioherbicide to Control Several Species of Pigweeds", in which a novel
Phomopsis sp. fungus is used as an effective broad-spectrum bioherbicide
for controlling pigweed.
The prior art teaches that fungi developed as biological herbicides should
be restricted in host range to one or a limited number of closely related
plant species. M. verrucaria, when manipulated as described, exhibits
herbicidal activity for a much wider range of plant species in numerous
plant families than has been previously reported.
Previous research has shown that not all plant pathogens are suitable
candidates for manipulation as biological herbicides. Indeed, of the
hundreds of fungi studied as potential biological herbicides, only a very
small number have been developed as biological herbicides. There are no
previous reports of any attempts to use any Myrothecium species as
biological herbicides. M. verrucaria under ordinary circumstances is only
weakly pathogenic. The process that we developed to manipulate this fungus
provides for a dramatic increase in pathogenic activity and increases the
host range to include many plant species not previously reported to be
hosts for the fungus. The host range of this fungus could not have been
predicted.
Pathogenic activity is enhanced by unusually high numbers of spores that
are applied in a suitable surfactant. As the research results herein
indicate, large numbers of spores applied in water only, will not produce
the desired level of disease activity. The type of surfactant also
influences disease activity. The use of nonoxynol (9 to 10 POE)
›a-(p-nonylphenyl)-w-hydroxypoly (oxyethylene)! surfactant, marketed as
STEROX N.J. (trademark) and the organosilicone surfactant SILWET L-77
(trademark) results in significant increases in disease activity. Inoculum
concentration has been shown to be important in the performance of
potential biological herbicides, but the dramatic increases in disease
activity with increased inoculum levels could not have been predicted.
Prior art also teaches that surfactant selection can provide increased
levels of activity, but the dramatic increases in disease activity and
host range documented herein for this fungus could not have been predicted
based on prior art.
The responses of closely related species of plants can vary greatly from no
symptoms for some species to severe stunting or death for other species.
These differential responses of closely related species could not have
been predicted. Combinations of A. cassiae and M. verrucaria exhibited
synergistic activity that could not have been predicted. Many organisms
used in combination exhibit antagonistic activity toward each other,
resulting in lower levels of control than would be expected from either
pathogen alone. Results indicating that combinations of A. cassiae and M.
verrucaria are synergistic could not have been predicted. This is
especially true in view of our observations and reports by others that M.
verrucaria inhibits the growth in vitro of a number of fungi, including
Alternaria spp.
The selectivity that can be obtained by the method of application could not
have been predicted based on prior art. Test results indicate the
potential for using directed spray applications to control susceptible
weed species growing beneath crop canopies. When spores plus certain
surfactants are applied over the top of the foliage of some important crop
species, such as soybeans or cotton, many of the plants are killed, as
tabulated in Table I, herein. However, it was observed that when the same
spore suspensions are applied below the leaves, these crop species were
not adversely affected.
Myrothecium verrucaria is cosmopolitan and there are numerous reports in
the literature related to this species. This organism is usually
considered to be a soil inhabiting organism that is nonpathogenic or
weakly pathogenic. No reports of disease injury caused by this organism
document results that would suggest possible use of M. verrucaria as a
microbial herbicide.
The invention set forth in this application is based on a combination of
factors that include the particular isolate of M. verrucaria, inoculum
concentration, and the use of a suitable surfactant. The dramatic
interactions that the test data indicate could not have been predicted
from the prior art.
Effective inoculum concentrations, when applied in water only, result in
levels of efficacy that are too low for use as a biological herbicide.
No other potential microbial herbicide reported in the scientific
literature maintained herbicidal activity at 35.degree. C. with no
apparent reduction in efficacy. Indeed, the herbicidal activity of this
invention appeared to increase with temperature, as indicated by the
number of plants that exhibited collapse of stem and leaf tissue
immediately following a 6 hour dew period at 35.degree. C.
Prior art teaches that fungi applied as microbial herbicides to the foliage
of target plants require dew periods, typically 6 to 18 hours, for desired
efficacy. There are numerous reports of special formulations involving
invert emulsions, oils, or hydrophilic ingredients to circumvent or
supplement requirements for moisture. Our invention provides a control
which is not dependent upon these formulations.
SUMMARY OF THE INVENTION
The present invention is directed to a method for biological control of
various weeds, including sicklepod, pigweed, spurred anoda, jimsonweed,
hemp sesbania, and other susceptible weeds using the fungus Myrothecium
verrucaria. The fungus is initially isolated from diseased plants and then
conidia are produced during incubation in petri dish cultures. In one
embodiment inoculum is produced by harvesting conidia from the petri
dishes using distilled water or a surfactant in distilled water. In
another embodiment the harvested conidia are rinsed in distilled water
through repeated cycles of centrifugation. Resulting cell-free filtrates
containing phytotoxin rinsed from the fungus are applied to the weeds,
either alone or in combination with conidia of the fungus Alternaria
cassiae, to enhance pathogenic action of the fungus. In still another
embodiment inoculum is produced by combining conidia of M. verrucaria and
those of A. cassiae in a surfactant, to synergistically enhance pathogenic
action of A. cassiae.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will be better understood by reference to the following
drawings, wherein:
FIG. 1 is a graph of Myrothecium verrucaria inoculum concentration
(conidia) vs. mortality (%) of sicklepod;
FIG. 2 is a graph of dew period (hr) vs. reduction (%) of sicklepod by
Myrothecium verrucaria;
FIG. 3 is a graph of dew period temperature (.degree. C.) vs. reduction (%)
of sicklepod by Myrothecium verrucaria;
FIG. 4A is a graph of SILWET L-77 (trademark) surfactant concentration (%)
on Myrothecium verrucaria vs. sicklepod mortality (%); and
FIG. 4B is a graph of SILWET L-77 (trademark) surfactant concentration (%)
vs. sicklepod dry weight (G);
FIG. 5 is a graph of selected surfactants vs. reduction (%) of sicklepod;
FIG. 6 is a graph of stage of growth (leaf no.) of sicklepod seedlings vs.
reduction (%) of sicklepod; and
FIG. 7 is a bar graph of dew period (hr) vs. reduction (%) of sicklepod.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Myrothecium verrucaria (Alb. and Schwein) Ditmar ex Fr. is on deposit with
the Department of Biological Sciences, Louisiana Tech University in
Ruston, La. and was placed on deposit with the International Mycological
Institute, Bakeham Lane, Egham Surrey, UK on May 18, 1994, as IMI number
361690. The deposit was placed in the patent collection of the
International Mycological Institute and has been assigned the accession
number IMI 368023. The date of deposit was Jun. 21, 1995. The address of
the Department of Biological Sciences is: Louisiana Tech University,
Harrell L. Walker, Department of Biological Sciences, P.O. Box 3179, TS.,
Ruston, La. 71272.
According to (Tulloch, M. 1972, The Genus Myrothecium Tode ex Fr.
Mycological Papers 130:1-42), M. verrucaria is described as follows:
"Spore mass wet, black, convex surrounded by wet floccose margin. Spores
broadly fusiform, one end pointed the other protruding and truncate, in
erythrosin and NH4 solution with a fantailed appendage on the pointed end,
6.5-8.times.2.3-5 microns."
The Myrothecium verrucaria used in this invention was isolated from
diseased plants of sicklepod, a new host record for this fungus. Unless
otherwise stated, the fungus was cultured on potato dextrose agar (PDA) in
petri dishes that were inverted and placed on open-mesh, wire shelves of a
Percival model I 35 LLVL incubator at 24.degree. C. Twelve-hour
photoperiods were provided by two, 20-watt cool-white fluorescent lights
directed upwardly from 10 cm. below the cultures. Colonies produced
concentric rings of spores (conidia) under these culture conditions and
the spores were harvested by rinsing the cultures with 0.05% surfactant.
Inoculum was prepared by suspending the conidia in 0.05% surfactant and
adjusted to a concentration of 20.times.10.sup.6 conidia per ml, using a
hemacytometer.
Unless otherwise indicated, experimental units consisted of groups of 12
plants that were grown in peat strips of 12, 5.8 cm square pots. The
growing medium consisted of a commercial blend of peat and perlite that
was supplemented with controlled release 13--13--13 fertilizer. Plants
were sprayed to wetness with foliar applications of spore suspensions
containing 20.times.10.sup.6 conidia per ml and 0.05% (v/v) SILWET L-77
(trademark), a silicone-polyether copolymer spray adjuvant. Control groups
were sprayed with 0.05% surfactant only. Aerosol sprayers were used to
make the applications and treatments were replicated three times.
Conidia of M. verrucaria were harvested from cultures grown on PDA and
rinsed three times in distilled water by sequential centrifugation cycles
at 840.times. g for 10 minutes per cycle. A cell-free filtrate was
prepared by filtering the supernatant from the first centrifugation cycle
through 0.2 micron membrane filters. The washed conidia produced only mild
disease symptoms when inoculated to host plants, indicating an important
role of phytotoxin in disease development. The cell-free filtrate
containing phytotoxin resulted in a decrease in dry weight, but minimum
rates of mortality, when applied to sicklepod.
Conidia of M. verrucaria and conidia of A. cassiae were harvested from
cultures grown on PDA and vegetable juice agar, respectively, suspended in
0.05% surfactant, to a concentration of 5.times.10.sup.4 A. cassiae
conidia per ml and 1.times.10.sup.7 M. verrucaria conidia per ml. The
mixture was subsequently applied to test plants. Combinations of A.
cassiae and M. verrucaria surprisingly exhibited a synergistic effect on
the test plants, resulting in a considerably greater kill rate of the
plants than was achieved using either fungus alone.
Conidia of M. verrucaria were harvested from cultures grown on PDA and
rinsed once in distilled water by a centrifugation cycle at 840.times. g
for 10 minutes. The M. verrucaria conidia were discarded and a cell-free
filtrate was prepared by filtering the supernatant through 0.2 micron
membrane filters. Alternaria cassiae conidia were suspended in the
cell-free filtrate, which was subsequently applied to test plants.
Combinations of A. cassiae and cell-free filtrate derived from M.
verrucaria conidia surprisingly exhibited a synergistic effect on the test
plants, resulting in a considerably greater kill rate of the plants than
was achieved using either A. cassiae or cell-free filtrate alone.
A number of plant species, representing a number of different families,
were tested for reaction to foliar applications of the fungus and the
results were tabulated in Table I. Test plants in the cotyledon to second
leaf stage of growth were inoculated and placed in unlighted dew chambers
for six hours at 25.degree. C., then moved to greenhouse benches and
observed for disease development. Results indicate that M. verrucaria,
when manipulated according to the method of the invention, has a broad
host range that includes plant species in a number of families. A search
of the literature indicated that many of these species apparently are new
hosts for M. verrucaria, and these species are also indicated in Table I.
It was surprisingly found that most of the grasses and sedges tested were
much more tolerant of the fungal preparation than were most of the
broadleaved species tested. Among the broadleaved species, unexpected
differences in susceptibilities were found. For example, sicklepod was
highly susceptible to the fungus, while coffee senna, a closely-related
species in the same genus, was much more tolerant. Velvetleaf was highly
susceptible, yet prickly sida, a species in the same family, was much more
tolerant. The data presented in Table I document these and other examples
of differences in responses by closely-related species.
TABLE 1
__________________________________________________________________________
HOST RANGE RESPONSE TO MYROTHECIUM VERRUCARIA
FAMILY COMMON MORTALITY (%)
MORTALITY (%)
DRY WEIGHT
SCIENTIFIC NAME
NAME 7 DAYS 14 DAYS REDUCTION (%)
NEW HOST
__________________________________________________________________________
RECORD
AMARANTHACEAE
Amaranthus retroflexus L.
Pigweed, redroot
78 81 89 YES
CHENOPODIACEAE
Beta vulgaris L.
Beet 100 100 94 YES
`Detroit Dark Red`
Chenopodium amaranticolor
Chenopodium
89 92 90 YES
Coste & Reynier
COMPOSITAE
Tagetes sp. Marigold 6 8 80 YES
`Petite Yellow`
Helianthus annuus L.
Sunflower 42 83 86 YES
`Mammoth Gray
Stripe`
.sup.1 Xanthium pensylvanicum
Cocklebur 0 17 91
Wallr.
Zinnia elegans Jacq.
Zinnia 14 75 90 YES
`Sombrero`
CONVOLVULACEAE
Ipomea spp. morning glory
0 0 35
CRUCIFEREAE
Brassica rapa L.
Turnip 33 47 83 YES
`Seven Top`
Raphanus sativus L.
Radish 100 100 93 YES
`Cherry Belle`
CUCURBITACEAE
Cucumis melo L.
Cantaloupe
0 0 73
`Hale's Best Jumbo`
Cucumis sativus L.
Cucumber 19 22 54
`Straight 8`
Curcubita pepo L.
Pumpkin 25 33 70
`Jack-O'-Lantern`
Cucurbita pepo var.
Squash 0 0 43
melopepo (L.) Alef.
`Yellow Crookneck`
Citrullus vulgaris Schrad.
Watermelon
0 0 49
`Charleston Gray`
CYPERACEAE
.sup.2 Cyperus rotundus L.
Nutsedge, purple
0 0 0
GRAMINEAE
.sup.3 Brachiaria platyphylla
Signalgrass, broadleaf
0 0 7
(Griseb.) Nash
.sup.4 Cynodon dactylon (L.)
Bermudagrass
0 0 30
Pers. `Tifway 328`
.sup.3 Digitaria ciliaris (Retz.)
Crabgrass, southern
0 0 14
Koel.
.sup.3 Echinochloa crus-galli (L.)
Barnyardgrass
0 0 20
Beauv.
.sup.4 Eremochloa ophiuroides
Centipedegrass
0 0 22
(Munro) Hack.
.sup.3 Leptochloa sp.
Sprangletop, tighthead
0 0 60
.sup.3 Poa annua L.
Bluegrass, annual
0 0 26
.sup.3 Setaria viridis (L.) Beauv.
Foxtail, green
0 0 28
Sorghum bicolor (L.)
Sorghum, grain
0 0 4
Moench `Delta Pine G522DR`
.sup.3 Sorghum halepense (L.)
Johnsongrass
0 0 45
Pers.
.sup.4 Stenotaphrum secondatum
St. Augustinegrass
0 0 0
(Walt) Ktze.
Zea mays L. Corn 0 0 0
`Trucker's Favorite`
.sup.4 Zoysia sp.
Zoysiagrass
0 0 1
LEGUMINOSAE
Arachis hypogoea L.
Peanut
`Improved Spanish`
0 0 15
Cassia obtusifolia L.
Sicklepod 97 97 96 YES
Cassia occidentalis L.
Senna, coffee
0 0 50 YES
Glycine max (L.) Merr.
Soybean
`Braxton` 0 0 67
`Cajun` 0 0 65
`Crawford`
0 0 66
`Forrest` 0 0 75
Phaseolus vulgaris L.
Bean, garden
`Kentucky Wonder`
0 0 79
`Henderson Bush`
0 0 76
Pisum sativum L.
Pea, English
56 89 72
`Early Alaskan`
Sesbania exaltata (Raf.)
Sesbania, hemp
100 100 90 YES
Cory
Vigna sinensis (Torner)
Cowpea 0 0 87 YES
Savi. `California Pinkeye
Purple Hull`
MALVACEAE
Abelmoschus esculentus
Okra 0 0 53
(L.) Moench `Clemson Spineless`
Abutilon theophrasti
Velvetleaf
0 9 55 YES
Medic.
Anoda cristata (L.)
Anoda, spurred
64 69 90 YES
Schlecht.
Gossypium hirsutum L.
Cotton
`Stoneville 453`
0 0 84
`Stoneville 506`
0 0 81
Sida spinosa L.
Sida, prickly
0 0 53 YES
SOLANACEAE
Capsicum frutescens L.
Pepper, green
0 0 0
`California Wonder`
Datura stramonium L.
Jimsonweed
100 100 97 YES
Lycopersicon esculentum
Tomato 0 0 26
Mill. `Marglobe`
Solanum ptycanthum Dun.
Nightshade, eastern
0 0 37
black
.sup.5 Solanum tuberosum L.
Potato
`Kennebec`
0 0 16
`Krantz` 0 0 31
`Red LaSoda`
0 0 3
__________________________________________________________________________
.sup.1 Grown from seed, one plant per 7.6 cm pot. Each replication
consisted of plants in four pots.
.sup.2 Grown from field plants, 2-4 plants per 7.6 cm pot, and each
replication consisted of plants in one pot.
.sup.3 Seeds were broadcast in 5.7 cm peat pots, twelve pots per strip.
Plants were thinned to 5-10 seedlings per pot.
.sup.4 Centipedegrass, bermudagrass, St. Augustinegrass, and zoysiagrass
were grown in 7.6 cm pots, and each replication consisted of plants in on
pot.
.sup.5 Grown from tubers, two plants per 15.2 cm pot, and each replicatio
consisted of plants in one pot.
The following examples illustrate application of the fungus Myrothecium
verrucaria in controlling weeds:
EXAMPLE 1
Effect of Inoculum Concentration on Mortality of Sicklepod Seedlings:
Sicklepod seedlings were inoculated with suspensions containing 0, 5, 10,
or 20.times.10.sup.6 conidia per ml and surfactant according to the
procedure outlined above. The most effective inoculum concentration for
control of sicklepod under these conditions was in the range of
20.times.10.sup.6 conidia per ml, as illustrated in FIG. 1.
EXAMPLE 2
Effect of Dew Duration on Dry Weight and Mortality of Sicklepod Seedlings:
Sicklepod seedlings were inoculated and placed in dew chambers as described
in the above-delineated procedure. After 0, 4, and 6 hours, inoculated and
control plants were removed from the dew chambers, placed on greenhouse
benches, and observed for disease development. A dew period of 6 hours
resulted in 85% kill of sicklepod seedlings 14 days after inoculation, as
illustrated in FIG. 2. At an inoculum level of 20.times.10.sup.6 spores
per ml there was a 60% reduction in dry weight with no dew following
inoculation.
EXAMPLE 3
Effect of Dew Period Temperature on Dry Weight and Mortality of Sicklepod
Seedlings:
Sicklepod seedlings were inoculated with 20.times.10.sup.6 conidia per ml
and placed in dew chambers at 10.degree., 15.degree., 20.degree.,
25.degree., 30.degree., and 35.degree. C. for 6 hours according to the
procedure described above. Control plants were sprayed with surfactant
only and were included for each temperature. Up to 100% kill of sicklepod
seedlings was consistently observed with dew temperatures of 25.degree. C.
to 35.degree. C., as illustrated in FIG. 3. The rate of expression of
disease severity increased with temperature up to the maximum 35.degree.
C. tested. Collapse of leaf and stem tissue was often noted as inoculated
plants were removed from the dew chambers following a 6 hour dew period at
35.degree. C.
No other potential microbial herbicide ever reported in the scientific
literature maintained herbicidal activity at 35.degree. C. with no
apparent reduction in efficacy. Indeed, the herbicidal activity of our
invention appeared to increase with temperature, as indicated by the
number of plants that exhibited collapse of stem and leaf tissue
immediately following a 6 hour dew period at 35.degree. C.
EXAMPLE 4
Effect of various concentrations of SILWET L-77 (trademark) on Mortality
and Dry Weight of Sicklepod Seedlings
The dramatic increase in mortality of sicklepod with the addition of SILWET
L-77 (trademark) in increasing concentrations is illustrated in FIG. 4A.
Maximum sicklepod kill was realized at a SILWET L-77 (trademark)
concentration of 0.075%. Limited sicklepod mortality was noted in the
SILWET L-77 (trademark) application as a control without the fungus at
surfactant concentration of from 0.2 to about 0.4%.
FIG. 4B shows that under circumstances where the SILWET L-77 (trademark)
concentration exceeds 0.2% to 0.4% of the surfactant only, without the
fungus, as a control application, then there is a reduction in sicklepod
dry weight.
EXAMPLE 5
Effect of Different Surfactants on Mortality of Sicklepod Seedlings:
Spore suspensions containing 20.times.10.sup.5 conidia per ml were prepared
in distilled water only, 0.05% (w/v) oxysorbic (20 POE) polyoxyethylene
sorbitan mono oleate (TWEEN 80) (trademark) surfactant, 0.02% (v/v)
nonoxynol (9 to 10 POE) ›.alpha.-(p-nonylphenyl)-.omega.-hydroxypoly
(oxyethylene)! (STEROX NJ) (trademark) surfactant, and 0.05% (w/v) of
SILWET L-77 (trademark), as heretofore described. Sicklepod seedlings were
inoculated, placed in dew chambers at 25.degree. C. for 6 hours and moved
to greenhouse benches, as previously described. Control plants were
sprayed with water or surfactant only.
Seedling mortality was recorded 14 days after inoculation, and these data
are presented in FIG. 5. As indicated in FIG. 5, only 11% of the plants
were killed when the spores were suspended in water only, whereas 94% of
the plants were killed when the spores were suspended in SILWET L-77
(trademark) or STEROX NJ (trademark). The treatment utilizing the TWEEN 80
(trademark) surfactant was intermediate, resulting in 83% mortality.
EXAMPLE 6
Sicklepod Dry Weight and Mortality as Influenced by Stage of Growth of
Sicklepod Plants at Time of Inoculation:
Sicklepod seedlings in the cotyledonary to sixth true-leaf stage of growth
were inoculated, placed in dew chambers for 6 hours, then moved to
greenhouse benches, as previously described. Data for dry weight and
mortality 14 days after inoculation are presented in FIG. 6.
EXAMPLE 7
Increased Inoculum Levels Reduce the Dew Requirement:
As reported for many other pathogens, we observed an interaction of several
variables, particularly spore concentration, length of dew period, growth
stage of plants and surfactants. Other factors can also be important, but
manipulation of each of these parameters can significantly affect results.
For example, by extending the dew period from 6 hours to 18 hours,
mortality of sicklepod plants can be increased with the same inoculum
concentration. Also, by extending dew periods and increasing inoculum
levels, larger sicklepod plants can be controlled. An inoculum level of
4.times.10.sup.7 conidia per ml, suspended in 0.075% SILWET L-77
(trademark) resulted in significant mortality of sicklepod seedlings with
no dew, as illustrated in FIG. 7.
EXAMPLE 8
Efficacy of M. verrucaria as Affected by Rinsing of Spores:
A number of reports indicate phytotoxicity associated with sporodochial
fluids from petri dish cultures and metabolites produced in submerged
liquid cultures.
Conidia of M. verrucaria harvested from cultures grown on PDA as previously
described, were rinsed three times in distilled water by sequential
centrifugation cycles. These washed conidia that were rinsed using
repeated cycles of centrifugation did not produce any mortality or dry
weight reduction when inoculated to host plants, indicating an important
role of phytotoxin in disease development. Cell-free filtrates containing
phytotoxin when applied to sicklepod resulted in a decrease in dry weight,
but minimum rates of mortality, as detailed in the following Table 2.
TABLE 2
__________________________________________________________________________
SICKLEPOD DRY WEIGHT AND MORTALITY AS AFFECTED BY
RINSING OF MYROTHECIUM VERRUCARIA CONIDIA
DRY-WEIGHT
TREATMENT.sup.1 DRY WEIGHT (g)
REDUCTION (%)
MORTALITY (%)
__________________________________________________________________________
Control.sup.2 3.7 0.0 0.0
4 .times. 10.sup.7 Conidia Per Milliliter.sup.3
0.3 92.6 83.3
4 .times. 10.sup.7 Conidia Per Milliliter (Rinsed).sup.4
3.9 -5.5 0.0
Cell-Free Filtrate.sup.5
0.5 86.1 2.8
__________________________________________________________________________
.sup.1 Sicklepod plants in the cotyledonary to firsttrue leaf stage of
growth were sprayed to wetness with each treatment. The plants were place
in a dew chamber at 25 C for 6 hours, then moved to a greenhouse bench.
Plants were excised at the soil line 14 days after inoculation and dried
days at 75 C. Data are averages of three replications, 12 plants each.
.sup.2 Silwet L77 surfactant (0.5%).
.sup.3 Conidia were harvested in .05% surfactant by rinsing cultures grow
on potato dextrose agar.
.sup.4 Rinsed in distilled water by three centrifugation cycles at 840
.times. g for 10 minutes. After the third centifugation cycle, the conidi
were resuspended in .05% surfactant.
.sup.5 Supernatant from first centrifugation cycle of a suspension
containing 4 .times. 10.sup.7 conidia per milliliter in .05% surfactant
was filtered through 0.2.mu. membrane filters.
EXAMPLE 9
Combination of M. verrucaria and Alternaria cassiae for Control of
Sicklepod
Alternaria cassiae Juriar and Khan has been studied as a potential
biological control agent for sicklepod. Studies were conducted to
determine if sicklepod control could enhanced by combinations of A.
cassiae and M. verrucaria.
Sicklepod plants in the cotyledon to first leaf growth stage were
inoculated with concentrations of various combinations of A. cassiae and
M. verrucaria conidia that typically produce mortality rates of 50% or
less, as detailed in the following Table 3.
TABLE 3
______________________________________
SICKLEPOD MORTALITY AS AFFECTED BY COMBINATIONS OF
MYROTHECIUM VERRUCARIA AND ALTERNARIA CASSIAE
TREATMENT.sup.1 MORTALITY (%)
______________________________________
Control.sup.2 0.0
M. verrucaria.sup.3 0.0
M. verrucaria (rinsed spores).sup.4
0.0
Filtrate.sup.5 0.0
M. verrucaria (rinsed spores) + Filtrate.sup.6
0.0
A cassiae.sup.7 33.0
A. cassiae + M. verrucaria.sup.8
81.0
A. cassiae + Filtrate.sup.9
81.0
______________________________________
.sup.1 Sicklepod plants in the cotyledonary to first trueleaf stage of
growth were sprayed to wetness with each treatment. The plants were place
in a dew chamber at 25 C for 6 hours, then moved to a greenhouse bench.
Data, collected 14 days after treatment, are averages of three
replications, 12 plants each.
.sup.2 Silwet L77 surfactant (0.05%).
.sup.3 Inoculum contained 1 .times. 10.sup.7 conidia per milliliter in
surfactant (0.05%).
.sup.4 Conidia were harvested from cultures grown on potato dextrose agar
by rinsing with 0.05% surfactant. These conidia were rinsed three times i
distilled water by sequential centrifugation cycles at 840 .times. g for
10 minutes. After the third centrifugation cycle, the conidia were
resuspended in 0.05% surfactant.
.sup.5 A cellfree filtrate was prepared by filtering the supernatant from
the first centrifugation cycle through 0.2.mu. membrane filters.
.sup.6 Pellets of rinsed conidia from the third centrifugation cycle were
resuspended in cellfree filtrate so that the concentration of conidia was
1 .times. 10.sup.7 conidia per milliliter.
.sup.7 Alternaria cassiae inoculum contained 5 .times. 10.sup.4 conidia
per milliliter in 0.05% surfactant.
.sup.8 Alternaria cassiae inoculum (5 .times. 10.sup.4) plus Myrothecium
verrucaria inoculum (1 .times. 10.sup.7) (unrinsed).
.sup.9 Alternaria cassiae inoculum (5 .times. 10.sup.4 per milliliter)
suspended in cellfree filtrate prepared from Myrothecium verrucaria
conidia.
Results recorded after 14 days indicate rates of mortality when the two
pathogens were combined that exceeded the additive effects of the
pathogens (Table 3).
Combinations of A. cassiae and M. verrucaria exhibited synergistic activity
that could not have been predicted. Many organisms used in combination
exhibit antagonistic activity toward each other. Results indicating that
A. cassiae and M. verrucaria can be used in combination could not have
been predicted. This is especially true in view of our observations and
reports by others that M. verrucaria inhibits the growth of a number of
fungi, including Alternaria spp.
The experimental parameters used in examples cited for this invention are
not intended to limit the scope of this invention. Modification of factors
such as inoculum concentrations, parameters for inoculum production,
surfactants, application methods, and other factors, would be expected to
influence efficacy of this invention. Parameters were selected to enable
detection of interactions, to document the relationship of this invention
to the prior art, and to illustrate that the unique and surprising
characteristics of this invention were not obvious and could not have been
predicted from the prior art.
While we recognize that various plant species will exhibit some differences
in response to this pathogen, the scope of this invention relates to the
use of M. verrucaria as a broad-spectrum biological herbicide. As
indicated in Table I, sicklepod is only one example of numerous weed
species that are affected by M. verrucaria. Examples utilizing sicklepod
as the target species are not intended to limit the scope of this
invention.
While the preferred embodiments have been described above, it will be
recognized and understood that various modifications may be made in the
invention and the appended claims are intended to cover all such
modifications which may fall within the spirit and scope of the invention.
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